Abstract

It has been reported that WIN55212-2, a prototypic aminoalkylindole, has higher affinity for CB₂ than for CB₁. To explain the selectivity of WIN55212-2 for CB₂, molecular modeling studies were performed to probe the interacting sites between WIN55212-2 and cannabinoid receptors. In TMH5 the position 5.46 is a Phe in CB₂ versus a Val in CB₁. Docking of WIN55212-2 into the models of CB₁ and CB₂predicts that F5.46 will result in a greater aromatic stacking of CB₂ with WIN55212-2. Using site-directed mutagenesis, this hypothesis was tested by exchanging the amino acids at position 5.46 between CB₁ and CB₂. Two mutations, including a Phe to Val mutation at the position 5.46 in CB₂(CB2F5.46V), and a corresponding Val to Phe mutation at the position 5.46 in CB₁ (CB₁V5.46F), were made. The mutant receptors were transfected into 293 cells, and stable cell lines expressing similar numbers of receptors as wild-type receptors were chosen for additional ligand binding and cAMP accumulation studies. In ligand- binding assays, the CB₂F5.46V mutation decreased the affinity of WIN55212-2 for CB₂ by 14-fold. In contrast, the CB₁V5.46F mutation increased the affinity of WIN55212-2 for CB₁ by 12-fold. However, these mutations did not change the affinity of HU-210, CP-55940, and anandamide for CB₁and CB₂. In cAMP accumulation assays, the changes in EC₅₀ values of WIN55212-2 were consistent with the changes in its binding affinity caused by the mutations. These results strongly support the hypothesis that the selectivity of WIN55212-2 for CB₂ over CB₁ is attributable to the change from Val in CB₁ at position 5.46 to Phe in CB₂.